The 2014 outbreak in West Africa, cause by Ebola virus (EBOV), has highlighted the global threat of filoviruses. Major progress has been made recently towards vaccines and monoclonal antibody (MAb) therapeutics specific for EBOV including development of ZMapp, a cocktail of three mAbs, and two vaccine platforms that advanced to clinical trials. The success of ZMapp has highlighted the critical role of antibodies in protecting against filovirus hemorrhagic fever. However, cross reactive MAbs and vaccines that would also protect against other members of Filoviridae such as Marburg, Sudan, and Bundibugyo viruses are lagging behind. Little is known about the key features of broadly protective responses including cross-species neutralizing epitopes within filovirus surface glycoprotein (GP), the mechanisms by which antibodies against different epitopes synergize for effective neutralization, features that determine the evolution of cross protective versus species specific B cell responses, and the extent to which neutralizing and cross-reactive antibodies are elicited after vaccination or natural exposure. This proposal aims to address these important questions. Using a soluble HIV envelope glycoprotein as model antigen and a combination of B cell cloning and deep sequencing we have extensively characterized the features of broadly neutralizing HIV antibodies including the VH/VL usage and the degree of somatic hypermutations (SHM) in rhesus macaques. Here we intend to use a similar approach to understand the evolution of B cell responses to filovirus antigens in macaques. Cynomolgus and rhesus macaques are highly relevant models for filovirus vaccine development and, given the high homology between the human and monkey immunoglobulin VH loci (91.72.2%), also for the study of the B cell evolution or identification of therapeutic filovirus MAbs. We have recently performed two immunization studies using filovirus antigens in cynomolgus and rhesus macaques and generated a large collection of peripheral blood mononuclear cells (PBMC), bone marrow, and splenocytes that will be used in this proposal for in depth characterization of broadly protective B cell responses and identification of novel therapeutic MAbs. Using the PBMC from the first cynomolgus study we have already identified several pan-ebolavirus and pan-filovirus antibodies including MAbs that cross-neutralize and protect against SUDV and EBOV in mice and guinea pigs. This is the first report of antibodies protective against these two most divergent ebolavirus species. We have also discovered a novel mechanism of cooperative neutralization by certain pan-ebolavirus MAbs, a phenomenon that builds a rational basis for design of effective MAb cocktails as well as broadly protective vaccines. This proposal is designed in three specific Aims. In Aim 1, we will define the genetic elements of cynomolgus nave Ig repertoire to complement the knowledge that already exists on rhesus. Novel cross-reactive filovirus MAbs from cynomolgus and rhesus macaques will be isolated using antigen-specific memory B cell sorting and cloning. Using antibodyome analysis by NGS, we will define the features of broadly protective B cell responses including VH/VL gene usage and SHM. In Aim 2, the epitopes and mechanism of action of the novel cross reactive MAbs will be extensively analyzed and MAbs will be tested for in vivo efficacy. Novel concept will be developed and evaluated for immunogen improvement for pan-filovirus vaccines. In Aim 3, antibody responses to filovirus vaccines will be profiled in vaccinated macaques and in human samples from a clinical trial in West Africa (NewLink Genetics/Merck) as well as in a large number of sera from fatal and convalescent naturally infected humans. This multiple PI proposal will be led by Dr. M. Javad Aman and Dr. Yuxing Li with complementary expertise in filovirus vaccines and immunotherapeutics and immunogenetics of macaques. The team further includes Dr. John Dye with expertise in filovirus animal models, Dr. Kartik Chandran, an expert in filovirus receptor biology, Dr. Gary Kobinger, who led the development of ZMapp and was intimately involved in 2014 outbreak response in Sierra Leone, and Dr. Thomas Monath, NewLink Genetics, who will provide access to material from clinical trials of EBOV VSV vaccine in West Africa.